Overlapping DNA recognition motifs between Sp1 and a novel trans-acting factor within the wt1 tumour suppressor gene promoter

The Wilms' tumor suppressor gene, wt1 , encodes a zinc finger transcription factor which has been shown to regulate the expression of several genes involved in cellular proliferation and differentiation. Expression of wt1 is developmentally regulated and restricted to a small set of tissues which include the fetal urogenital system, mesothelium and spleen. A highly conserved motif within the wt1 promoter, located between nucleotides -34 and -71 relative to the first transcription start site in the murine promoter, harbors consensus binding sites for Sp1 and members of the paired-box transcription factor family. Pax-2 and Pax-8 are known to enhance expression of wt1 through this conserved regulatory element. In this report, we demonstrate that Sp1 is able to bind to two sites within the 38 bp conserved region (CR). By electrophoretic mobility shift assays (EMSAs), we have identified a novel binding activity, referred to as complex D, which recognizes sequences overlapping one of the Sp1 sites in the CR. EMSA competition experiments indicate that binding of complex D and Sp1 to the CR is mutually exclusive and Sp1 is able to displace complex D binding. In situ UV crosslinking and molecular mass determinations indicate that complex D is a complex of approximately 130 kDa, consisting of at least two proteins of approximately 62 and approximately 70 kDa. Transient transfections suggest that complex D may function as an activator.